PROJECT ABSTRACT Autophagy is a conserved, eukaryotic, cellular pathway responsible for the sequestration and degradation of intracellular macromolecules and macromolecular assemblies that are unnecessary, defective or harmful to cells. BECN homologs are highly conserved in eukaryotes, but mammals are unique in having two functional BECN paralogs. Defects or deficiencies in BECN1-mediated autophagy are implicated in a wide range of diseases such as breast and ovarian cancer, cardiovascular diseases, embryonic defects or fatality, as well as several infectious diseases, especially those caused by viruses; while defects in BECN2 are implicated in obesity, insulin resistance and Kaposi's Sarcoma associated Herpesvirus induced oncogenicity. The interaction of a BECN protein with ATG14 is essential for autophagosome nucleation, the first committed step in autophagy. BECN and ATG14 are complex, multi-domain proteins. The interaction of ATG14 with the BECN paralogs is mediated by the coiled-coil domain (CCD) within each protein. The long-term goal of the proposed research is to understand the mechanism of BECN homologs in autophagy and cellular homeostasis. The objective of this proposal is to obtain comparative structural and mechanistic information about the CCDs of human BECN paralogs, and their interaction with ATG14, which is essential for their function in mediating autophagy via the autophagosome nucleation complex. This objective will be accomplished by the specific aims of this proposal, which are: (1) To elucidate similarities and differences between human BECN1 and BECN2 CCD homodimers. (2) To elucidate similarities and differences in the interactions of human BECN1 and BECN2 CCDs with the ATG14 CCD. A wide range of structural, biophysical, biochemical, cellular and molecular biology methods will be used to accomplish these aims. The results from this research will lead to a mechanistic understanding of the competitive recruitment of ATG14 by the BECN paralogs, thereby providing invaluable insights into their different biological roles. This comparative understanding of the structural and thermodynamic details of how each paralog self assembles and recruits binding partners is essential to understanding autophagy and cellular homeostasis.